U.S. patent application number 12/750201 was filed with the patent office on 2011-08-25 for modulation of gene expression by oligomers targeted to chromosomal dna.
This patent application is currently assigned to Board of Regents, The University of Texas System. Invention is credited to David Reid Corey, Bethany Ann Janowski, John Dorrance Minna, David S. Shames.
Application Number | 20110207217 12/750201 |
Document ID | / |
Family ID | 38309687 |
Filed Date | 2011-08-25 |
United States Patent
Application |
20110207217 |
Kind Code |
A1 |
Corey; David Reid ; et
al. |
August 25, 2011 |
Modulation of Gene Expression by Oligomers Targeted to Chromosomal
DNA
Abstract
Synthesis of a target transcript of a gene is selectively
increased in a mammalian cell by contacting the cell with a
polynucleotide oligomer of 12-28 bases complementary to a region
within a target promoter of the gene under conditions whereby the
oligomer selectively increases synthesis of the target
transcript.
Inventors: |
Corey; David Reid; (Highland
Park, TX) ; Janowski; Bethany Ann; (Highland Park,
TX) ; Shames; David S.; (Dallas, TX) ; Minna;
John Dorrance; (Dallas, TX) |
Assignee: |
Board of Regents, The University of
Texas System
|
Family ID: |
38309687 |
Appl. No.: |
12/750201 |
Filed: |
March 30, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11599566 |
Nov 13, 2006 |
7709456 |
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12750201 |
|
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60738103 |
Nov 17, 2005 |
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Current U.S.
Class: |
435/375 |
Current CPC
Class: |
C12N 15/1135 20130101;
C12N 15/1138 20130101; C12N 15/111 20130101; A61K 48/005 20130101;
C12N 2310/322 20130101; C12N 15/113 20130101; C12N 15/63 20130101;
C12N 2310/33 20130101; C12N 2310/315 20130101; C12N 2320/50
20130101; C12N 2310/321 20130101; C12N 2310/331 20130101; C12N
2310/321 20130101; C12N 2310/3521 20130101 |
Class at
Publication: |
435/375 |
International
Class: |
C12N 5/02 20060101
C12N005/02 |
Goverment Interests
[0002] This work was made with Government support under grants
awarded by the National Institutes of Health (NIGMS 60642 and
73042; and P50 CA 70907). The Government has certain rights in this
invention.
Claims
1. A method of selectively increasing synthesis of a target
transcript of a gene in a mammalian cell, wherein the target
transcript is predetermined to be in need of increased synthesis,
the method comprising the steps of: contacting the cell with a
polynucleotide oligomer of 12-28 bases complementary to a region
within a target promoter of the gene under conditions whereby the
oligomer selectively increases synthesis of the target transcript;
and detecting resultant selective increased synthesis of the target
gene; wherein the oligomer is double-stranded RNA, and wherein the
region is located between nucleotides -100 to +25 relative to a
transcription start site of the gene.
2. The method of claim 1, wherein the region is located between
nucleotides -50 to +25 relative to a transcription start site of
the gene.
3. The method of claim 1, wherein the region is located between
nucleotides -30 to +17 relative to a transcription start site of
the gene.
4. The method of claim h wherein the region includes a
transcription start site of the gene.
5. The method of claim 1, wherein the target promoter is the
promoter of the target transcript.
6. The method of claim 1, wherein the target promoter is the
promoter of an isoform of the target transcript.
7. The method of claim 1, wherein the target promoter is the
promoter of an predetermined isoform of the target transcript, and
synthesis of the isoform is inhibited.
8. The method of claim 1, wherein the target promoter is both the
promoter of the target transcript and the promoter of an isoform of
the target transcript.
9. The method of claim 1, wherein the oligomer is double-stranded
RNA of 18-25 bases.
10. The method of claim 1, wherein the oligomer comprises a
nucleotide having a 2' chemical modification.
11. The method of claim 1, wherein the oligomer comprises a serum
stability-enhancing chemical modification selected from the group
consisting of a phosphorothioate internucleotide linkage, a
2'-O-methyl ribonucleotide, a 2'-deoxy-2'-fluoro ribonucleotide, a
2' deoxy ribonucleotide, a universal base nucleotide, a 5-C-methyl
nucleotide, an inverted deoxyabasic residue incorporation, and a
locked nucleic acid.
12. The method of claim h wherein the cell is a cultured cell in
vitro.
13. The method of claim 1, wherein the cell is in situ in a
host.
14. The method of claim 1, wherein the contacting step is free of
viral transduction.
15. The method of claim 1, wherein the contacting step is free of
viral transduction, and the cell is contacted with a composition
consisting essentially of the oligomer.
16. The method of claim 1, wherein the contacting step is free of
viral transduction, and there is at least a 2-fold resultant
increased synthesis of the target transcript.
17. The method of claim 1, wherein the oligomer is a
double-stranded RNA of 18-25 bases, a single region of the target
promoter is targeted, and there is at least a 2-fold resultant
increased synthesis of the target transcript.
18. The method of claim h wherein the cell is contacted with a
1-100 nanomolar concentration of the oligomer.
19. The method of claim 1, wherein the cell is a cancer cell and
the gene encodes a protein selected from the group consisting of
E-cadherin, human progesterone receptor (hPR), p53, and PTEN.
20. A method of decreasing Cox-2 expression in a cell, the method
comprising the steps of: contacting the cell with polynucleotide
oligomer of 12-28 bases complementary to a region located between
nucleotides -100 to +25 relative to a transcription start site of a
human progesterone receptor (hPR) gene under conditions whereby the
oligomer selectively increases synthesis of the hPR; and detecting
decreased synthesis of the Cox-2; wherein the oligomer is
double-stranded RNA.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 60/738,103 filed Nov. 17, 2005.
FIELD OF THE INVENTION
[0003] The field of the invention is modulating gene transcript
synthesis using polynucleotide oligomers targeting a promoter
region of the gene.
BACKGROUND OF THE INVENTION
[0004] The ability of duplex RNA to recognize mRNA and silence gene
expression through post-transcriptional RNA interference (RNAi) is
widely appreciated (Tang, 2004). Short interfering RNAs (siRNAs)
have become common laboratory tools for controlling gene expression
and endogenously expressed microRNAs (miRNAs) participate in an
expanding array of cellular pathways.
[0005] RNA-directed DNA methylation was described originally in
plants (Matzke et al 2004), where it was found that RNA viruses and
viroids could induce methylation in genomic DNA sequences
(Massenegger et al 1994). Methylated bases were concentrated within
sequences of DNA that were complementary to RNA, suggesting a
sequence-specific mechanism for recognition (Pelissier and
Wassenegger 2000).
[0006] In yeast, small RNAs that target centromere repeat sequences
and mating type loci can silence gene expression by promoting
modification of heterochromatin (Grewal and Moazed 2003; Bernstein
and Allis 2005). Chromatin modifications involve methylation of
histone H3 at Lysine 9 (Volpe et al 2002) and require RNA-dependent
RNA polymerase (Sugiyarna et al 2005) and DNA polymerase II
(Schramke et al 2005). Modification involves proteins of the
RNA-induced transcriptional silencing (RITS) pathway (Verdel et al
2003) including argonaute 1 (Sigova et al 2004), a member of a
protein family that is also involved in post-transcriptional
silencing.
[0007] Recently, several reports have suggested that antigene RNAs
(agRNAs)--short oligonucleotides that target chromosomal DNA--can
also silence expression in mammalian cells. Kawasaki and Taira
targeted ten duplex RNAs to sequences within the E-cadherin
promoter that contained CpG dinucleotides (Kawasaki and Taira,
2004). DNA methylation was observed at all of these sites.
Individual RNAs yielded only marginal reductions in E-cadherin
expression but more complete silencing could be achieved if all ten
RNAs were combined. A link between methylation and silencing was
supported by the observation that duplex RNAs were not able to
inhibit expression of E-cadherin when methyl-transferase genes
DMNT1 and DMNT3B were silenced.
[0008] In a similar study, Morris and co-workers demonstrated that
duplex RNAs targeting the promoter of Elongation factor 1.alpha.
(EF1A) could inhibit expression (Morris et al, 2004). They observed
methylation of DNA at the target sequence and that addition of the
methylation inhibitor 5'-aza-2'-deoxycytidine (5-aza-dC) in
conjunction with the histone deacetylase inhibitor trichostatin
(TSA) reversed silencing. The studies from the Taira and Morris
laboratories were significant because they provided evidence that
RNA could target DNA for silencing in mammalian cells and suggested
that RNA could induce DNA methylation. In the Morris study
silencing by a synthetic agRNA required use of a peptide designed
to promote nuclear uptake, but other studies have suggested that
standard transfection procedures are adequate (Kawasaki and Taira
2004; Castanotto et al 2005; Janowski et al 2005; Ting et al
2005).
[0009] Other attempts to achieve RNA-directed methylation in
mammalian cells have been less successful. Steer and coworkers
tested RNAs that targeted the gene encoding Huntingtin and did not
detect any methylation (Park et al 2004). No RNA-directed
methylation was observed upon stable expression of double-stranded
RNA in mouse oocytes (Svoboda, P. et al 2004). Rossi and colleagues
used expressed short hairpin RNAs (shRNAs) to target a
well-characterized CpG island within the promoter for the tumor
suppressor RASSF1A (Castanotto et al 2005). They reported modest
inhibition of gene expression. The methylation-specific PCR assay
showed methylation, but the more complete bisulphite sequencing
assay did not.
[0010] Our laboratory discovered that efficient RNA-mediated
silencing of chromosomal DNA can be achieved independent of DNA
methylation (Janowski et al 2005; U.S. Pat appl No. 60/661,769). We
targeted transcription start sites to block expression by
obstructing the initiation of transcription. A practical advantage
of targeting transcription start sites is that they occur in all
genes and provide a general and predictable class of target
sequences; targeting transcription start sites would also be
expected to block gene expression regardless of whether methylation
occurs.
[0011] In contrast to other studies we observed no methylation by
methylation-specific PCR or sodium bisulfite sequencing. Inhibition
of methyl transferase activity using 5-azaC or an anti-methyl
transferase siRNA had no effect on gene silencing, suggesting that
methylation was not involved in silencing. The silencing we
observed was more potent than that reported in prior studies,
indicating that transcription start sites may be particularly
susceptible targets for agRNAs.
[0012] Baylin and colleagues revisited transcriptional silencing of
E-cadherin (Ting et al 2005). They observed efficient silencing of
gene expression when two promoter-targeted duplex RNAs were used in
tandem, but not when the RNAs were used individually. Baylin
observed no evidence for DNA methylation.
[0013] It has been reported that siRNAs targeting the E-cadherin
gene promoter can activate transcription (Li et al, 2005) in
cultured breast cancer cells. Similarly, data has been presented
indicating increased EF1A mRNA expression by promoter-targeted
siRNA (Morris et al 2004; see FIG. 3A, first two bars). It has also
been reported that nuclear localized small modulatory
double-stranded (ds) RNA (smRNA) coding NRSE sequences triggered
activation of transcription of NRSE genes in adult hippocampal
neural stem cells (Kuwabara et al, 2004; and Kuwabara et al,
2005).
SUMMARY OF THE INVENTION
[0014] One aspect of the invention is a method of selectively
increasing synthesis of a target transcript of a gene in a
mammalian cell, wherein the target transcript is predetermined to
be in need of increased synthesis, the method comprising the steps
of: contacting the cell with a polynucleotide oligomer of 12-28
bases complementary to a region within a target promoter of the
gene under conditions whereby the oligomer selectively increases
synthesis of the target transcript; and detecting resultant
selective increased synthesis of the target gene.
[0015] In one embodiment, the region is located between nucleotides
-100 to +25 relative to a transcription start site of the gene. In
further embodiments, the region is located between nucleotides -50
to +25, -30 to +17, and -15 to +10, relative to a transcription
start site of the gene. In a particular embodiment, the region
includes nucleotides -9 to +2 relative to a transcription start
site of the gene. In a particular embodiment, the region includes a
transcription start site of the gene.
[0016] In one embodiment, the target promoter is the promoter of
the target transcript. In another embodiment, the target promoter
is the promoter of an isoform of the target transcript. In further
embodiments, the target promoter is both the promoter of the target
transcript and the promoter of an isoform of the target
transcript.
[0017] In one embodiment, the oligomer is selected from the group
consisting of a double-stranded RNA, a DNA, a peptide nucleic acid,
and a morpholino. In a particular embodiment, the oligomer is a
double-stranded RNA of 18-25 bases.
[0018] In one embodiment, the oligomer comprises a nucleotide
having a 2' chemical modification. In particular embodiments the
oligomer comprises a serum stability-enhancing chemical
modification selected from the group consisting of a
phosphorothioate internucleotide linkage, a 2'-O-methyl
ribonucleotide, a 2'-deoxy-2'-fluoro ribonucleotide, a 2'-deoxy
ribonucleotide, a universal base nucleotide, a 5-C-methyl
nucleotide, an inverted deoxyabasic residue incorporation, and a
locked nucleic acid.
[0019] In one embodiment the cell is a cultured cell in vitro. In
other embodiments, the cell is in situ in a host.
[0020] In one embodiment, the contacting step is free of viral
transduction. In further embodiments, the contacting step is free
of viral transduction, and the cell is contacted with a composition
consisting essentially of the oligomer. In a further embodiment,
the contacting step is free of viral transduction, and there is at
least a 2-fold resultant increased synthesis of the target
transcript. In another embodiment, the oligomer is a
double-stranded RNA of 18-25 bases, a single region of the target
promoter is targeted, and there is at least a 2-fold resultant
increased synthesis of the target transcript. In another embodiment
the contacting step is free of viral transduction, and the oligomer
is not attached to a nuclear localization peptide.
[0021] In one embodiment, the cell is contacted with a 1-100
nanomolar concentration of the oligomer.
[0022] In one embodiment, the cell is a cancer cell and the gene
encodes a protein selected from the group consisting of E-cadherin,
human progesterone receptor (hPR), p53, and PTEN.
[0023] Another aspect of the invention is an isolated or synthetic
polynucleotide oligomer for selectively increasing synthesis of a
target transcript of a gene, the oligomer comprising a nucleotide
sequence of 12-28 bases complementary to a region within a target
promoter of the gene, wherein introduced into a cell comprising the
gene the oligomer selectively increases transcription of the target
transcript.
[0024] In one embodiment, the region is located between nucleotides
-100 to +25 relative to a transcription start site of the gene. In
further embodiments, the region is located between nucleotides -50
to +25, -30 to +17, and -15 to +10, relative to a transcription
start site of the gene. In a particular embodiment, the region
includes nucleotides -9 to +2 relative to a transcription start
site of the gene. In a particular embodiment, the region includes a
transcription start site of the gene.
[0025] In one embodiment, the target promoter is the promoter of
the target transcript. In another embodiment, the target promoter
is the promoter of an isoform of the target transcript. In further
embodiments, the target promoter is both the promoter of the target
transcript and the promoter of an isoform of the target
transcript.
[0026] In one embodiment, the oligomer is selected from the group
consisting of RNA, DNA, peptide nucleic acid, and morpholino.
[0027] In another embodiment, the oligomer is a double-stranded RNA
of 18-25 bases comprising a nucleotide sequence selected from the
group consisting of SEQ ID NO:1-11, and 12.
[0028] In particular embodiments, the target transcript encodes a
protein selected from the group consisting of human major vault
protein (MVP), human E-cadherin, human progesterone receptor (hPR),
human p53, and human PTEN. In one embodiment, the cell is a cancer
cell and the gene encodes a protein selected from the group
consisting of E-cadherin, human progesterone receptor (hPR), p53,
and PTEN.
[0029] A method of doing business comprising promoting, marketing,
selling or licensing a subject invention.
DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
[0030] The invention provides methods and compositions for
selectively increasing synthesis of a target transcript of a gene
in a mammalian cell, wherein the target transcript is predetermined
to be in need of increased synthesis, the method comprising the
steps of contacting the cell with a polynucleotide oligomer of
12-28 bases complementary to a region within a target promoter of
the gene under conditions whereby the oligomer selectively
increases synthesis of the target transcript; and detecting
resultant selective increased synthesis of the target gene.
[0031] The target transcript of the gene is predetermined to be in
need of increased synthesis using routine methods. For example,
reduced levels of a target transcript and/or protein relative to
desired levels may be directly measured. Alternatively, the need
for increased synthesis of a target transcript may be inferred from
a phenotype associated with reduced levels of the target
transcript.
[0032] In one embodiment, the region within the promoter of the
gene is selected from a partially single-stranded structure, a
non-B-DNA structure, an AT-rich sequence, a cruciform loop, a
G-quadruplex, a nuclease hypersensitive elements (NHE), and a
region located between nucleotides -100 to +25 relative to a
transcription start site of the gene.
[0033] Preferred AT-rich sequences are found in stretches of DNA
where local melting occurs, such as the promoters of genes where
protein machinery must gain access to single-stranded regions, and
preferably comprise the TATA box of the gene, and/or at least 60%
or 70% A+T.
[0034] Preferred cruciform structures are formed from palindromic
genomic sequences forming a hairpin structure on each strand,
wherein the repeated sequences are separated by a stretch of
non-palindromic DNA providing a single-stranded loop at the end of
each of the hairpins of the cruciform.
[0035] Preferred G-quadruplex structures are identified in promoter
regions of mammalian genes and are implicated in transcription
regulation. For example the nuclease hypersensitivity element III
of the c-MYC oncogene promoter is involved in controlling
transcription and comprises a pyrimidine-rich and purine-rich
sequences on the coding and noncoding strands, respectively, that
can adopt I-motif and G-quadruplex structures, respectively.
Stabilization of the G-quadruplex has been shown to lead to
repression of c-MYC (see e.g. Siddiqui-Jain, 2002).
[0036] In one embodiment, the region targeted is located between
nucleotides -100 to +25 relative to a transcription start site of
the gene. In certain preferred embodiments of the method, the
region is located on the template strand between nucleotides -30 to
+17 relative to a transcription start site of the gene. In another
embodiment, the region is located between nucleotides -15 to +10
relative to a transcription start site of the gene. In a further
embodiment, the region includes nucleotides -9 to +2 relative to a
transcription start site of the gene. In certain preferred
embodiments, the region includes a transcription start site of the
gene. In other embodiments, the region does not include any
sequence downstream from the transcription start, e.g. the sequence
is located between nucleotides -100 to +1. The oligomers used in
the subject invention target genomic sequence and not mRNA.
[0037] In certain embodiments, the gene is known to encode and/or
express one or more isoforms of the target transcript, and the
method of the invention selectively increases synthesis of the
target transcript over basal expression levels and/or control
condition levels, while synthesis of the isoform(s) of the target
transcript may decrease, increase, or stay the same. The target
transcript and the isoform(s) may share the same promoter and/or
transcription start site, or they may have different promoters
and/or transcription start sites. Accordingly, in various
embodiments, the target promoter is (1) the promoter of the target
transcript, (2) the promoter of an isoform of the target
transcript, or (3) is both the promoter of the target transcript
and the promoter of an isoform of the target transcript. Numerous
genes are known to express multiple isoforms; examples include p53
(Bourdon, 2005), PTEN (Sharrard and Maitland, 2000), Bcl-2-related
genes (Akgul, 2004), and survivin (Caldas et al, 2005). For
example, the methods can be used to increase expression of one
target transcript by directing oligomers to the transcription start
site of an isoform. Where synthesis of the target transcript is
increased, and synthesis of the isoform is inhibited, the method
effectively and selectively modulates relative isoform synthesis in
the host cell. Hence, increased synthesis of predetermined desirous
or underexpressed isoforms can be coupled with decreased synthesis
of predetermined undesirable or overexpressed isoforms. As
exemplified with p53.beta./p53 below, this embodiment can be used
to effect a predetermined isoform switch in the host cells.
[0038] The polynucleotide oligomer is of a sequence and length
sufficient to effect the requisite increase of target transcript
synthesis. As used herein, the Singular forms "a," "an," and "the,"
refer to both the singular as well as plural, unless the context
clearly indicates otherwise. For example, the term "an oligomer"
includes single or plural oligomers and can be considered
equivalent to the phrase "at least one oligomer." Suitable
oligomers are typically 12-28 bases in length, and are
complementary to a region within a target promoter of the gene
(i.e. Watson-Crick binding complementarity). The oligomer may
comprise any nucleic acid, modified nucleic acid, or nucleic acid
mimic that can recognize DNA by Watson-Crick base-pairing.
Mismatches between the oligomer and the region of the promoter
being targeted, particularly more than one mis-match, often
diminish the efficacy of increasing target transcript synthesis.
The oligomer may be single-stranded or double-stranded (i.e. a
duplex). In the case of duplex oligomers, a first strand is
complementary to the region of the promoter being targeted, and the
second strand is complementary to the first strand. The oligomer
may target homopyrimidine sequences, homopyrimidine sequences, or
mixed purine/pyrimidine sequences. A mixed purine/pyrimidine
sequence contains at least one purine (the rest being pyrimidines)
or at least one pyrimidine (the rest being purines). A variety of
oligomers are known in the art that are capable of Watson-Crick
base-pairing. In certain embodiments, the oligomer is selected from
a double-stranded RNA, a DNA, a peptide nucleic acid, and a
morpholino.
[0039] Double-stranded (ds) RNAs are particularly preferred
oligomers because they are relatively easy to synthesize, and have
been used in human clinical trials. Preferred dsRNAs have 18-25
bases complementary to the region of the promoter being targeted,
and optionally have 3' di- or trinucleotide overhangs on each
strand. Methods for preparing dsRNA and delivering them to cells
are well-known in the art (see e.g. Elbashir et al, 2001; WO/017164
to Tuschl et al; and U.S. Pat. No. 6,506,559 to Fire et al).
Custom-made dsRNAs are also commercially available (e.g. Ambion
Inc., Austin, Tex.). The dsRNA used in the method of the invention
may be chemically modified to enhance a desired property of the
molecule. A broad spectrum of chemical modifications can be made to
duplex RNA, without negatively impacting the ability of the dsRNA
to selectively increase synthesis of the target transcript. In one
embodiment, the dsRNA comprises one or more nucleotides having a 2'
modification, and may be entirely 2'-substituted. A variety of 2'
modifications are known in the art (see e.g. U.S. Pat. No.
5,859,221 to Cook et al.; U.S. Pat. No. 6,673,611 to ThompSon et
al; and Czauderna et al, 2003). A preferred chemical modification
enhances serum stability and increases the half-life of dsRNA when
administered in vivo. Examples of serum stability-enhancing
chemical modifications include phosphorothioate internucleotide
linkages, 2'-O-methyl ribonucleotides, 2'-deoxy-2'-fluoro
ribonucleotides, 2'-deoxy ribonucleotides, "universal base"
nucleotides, 5-C-methyl nucleotides, and inverted deoxyabasic
residue incorporation (see e.g. US Patent Publication No.
20050032733 to McSwiggen et al). The dsRNA may optionally contain
locked nucleic acids (LNAs) to improve stability and increase
nuclease resistance (see e.g. Elmen et al, 2005; and Braasch et al,
2003). Another type of modification is to attach a fluorescent
molecule to the oligomer, for example, TAMRA, FAM, Texas Red, etc.,
to enable the oligomer to be tracked upon delivery to a host or to
facilitate transfection efficiency determinations.
[0040] Methylase-dependent inhibition of transcription using
antigene dsRNA targeting CpG islands has been described (2, 3).
However, the method of the present invention is
methylase-independent, wherein synthesis of the target transcript
is increased independently of, and without requiring effective
methylation (e.g. transcript synthesis still occurs if the cell is
contacted with the oligomer in the presence of a methylase
inhibitor). In a particular embodiment of the invention, the target
region within the target promoter is not contained within a CpG
island. Algorithms for identifying CpG islands in genomic sequences
are known (e.g. see Takai and Jones, 2002; and Takai and Jones
2003). In another embodiment of the invention, the oligomer is a
double-stranded RNA, and the target region within the target
promoter does not include a CG dinucleotide.
[0041] Peptide nucleic acids (PNAs) are also preferred oligomers
for use in the method of the invention. Various PNA configurations
are known in the art. For example, the PNA oligomer may be
homopyrimidine, optionally prepared as a bisPNA, where one PNA
oligomer binds the target via Watson-Crick base pairing, and a
second oligomer binds via Hoogsteen recognition (see e.g. Nielsen,
2004); homopurine, optionally substituting one or more adenines
with diaminopurine (see e.g. Haaima et al, 1997); or mixed
purine/pyrimidine, optionally configured to form a tail-clamp at
the target sequence (see e.g. Kaihatsu et al, 2003). In a preferred
embodiment, the PNA is single-stranded mixed purine/pyrimidine.
[0042] DNA oligomers can also be used in the method of the
invention. However, unmodified oligodeoxynucleotides are subject to
rapid degradation by nucleases. Therefore, when DNA oligomers are
used, they preferably have chemical modifications to increase
nuclease resistance. A variety of chemical modifications to
increase nuclease resistance are known in the art. The simplest and
most widely used modification is the phosphorothioate (PS)
modification, in which a sulfur atom replaces a non-bridging oxygen
in the oligophosphate backbone. DNA oligomers are commercially
available through numerous vendors (e.g. Integrated DNA
Technologies, Coralville, Iowa).
[0043] Other types of oligomers that can be used include morpholino
oligomers (see e.g. Summerton and Weller, 1997) and LNAs (see e.g.
Wahlestedt et al, 2000).
[0044] The mammalian cell that is contacted with the oligomer can
be in vitro (e.g. a cultured cell), or in situ in a host. Examples
of cultured cells include primary cells, cancer cells (e.g. from
cell lines), adult or embryonic stem cells, neural cells,
fibroblasts, myocytes, etc. The cell can be from any mammal. In one
embodiment, the cell is a human cell in vitro. In a further
embodiment, the cell is a breast cancer cell and the gene is the
human progesterone receptor. In other embodiments, the cell is a
cancer cell and the gene encodes a protein selected from the group
consisting of E-cadherin, human progesterone receptor (hPR), p53,
and PTEN.
[0045] Cultured human cells commonly used to test putative
therapeutics for human diseases or disorders can be used to screen
oligomers that target promoter regions of genes for therapeutic
affect (e.g. induction of apoptosis, cessation of proliferation in
cancer cells, etc.). When the cell is in situ, the host may be any
mammal, and in certain preferred embodiments is a human, or an
animal model used in the study of human diseases or disorders (e.g.
rodent, canine, porcine, etc. animal models).
[0046] In the contacting step, the methods used to deliver the
oligomer to the cell can vary depending on the oligomer used and
whether the cell is in vitro or in vivo. For cells in vitro,
delivery can be accomplished by direct injection into cells. When
microinjection is not an option, delivery can be enhanced in some
cases by using hydrophobic or cationic carriers such as
Lipofectamine.TM. (Invitrogen, Carlsbad, Calif.). In one embodiment
of the invention, the cell is a cultured cell in vitro, the
oligomer is a double-stranded RNA of 18-25 bases, and the cell is
contacted with a composition comprising the oligomer and a cationic
lipid. PNA oligomers can be introduced into cells in vitro by
complexing them with partially complementary DNA oligonucleotides
and cationic lipid (21-25). The lipid promotes internalization of
the DNA, while the PNA enters as cargo and is subsequently
released. Peptides such as penetratin, transportan, Tat peptide,
nuclear localization signal (NLS), and others, can be attached to
the oligomer to promote cellular uptake (see e.g., Nielsen, 2004;
Kaihatsu et al, 2003; Kaihatsu, et al, 2004; and ref. 7).
Alternatively, the cells can be permeabilized with a
permeabilization agent such as lysolecithin, and then contacted
with the oligomer. Viral transduction can be used to deliver
oligomers to cells in vitro (e.g. lentiviral transduction, see e.g.
ref 7). However, in certain embodiments of the invention, it is
preferred that the contacting step is free of viral transduction.
In a further preferred embodiment, the contacting step is free of
viral transduction, and the oligomer is not attached to a nuclear
localization peptide.
[0047] For cells in situ, cationic lipids (see e.g. Hassani et al,
2004) and polymers such as polyethylenimine (see e.g. Urban-Klein,
2005) have been used to facilitate oligomer delivery. Compositions
consisting essentially of the oligomer (in a carrier solution) can
be directly injected into the host (see e.g. Tyler et al, 1999;
McMahon et al, 2002). In a preferred embodiment of the invention,
the cell is in situ in a host, the oligomer is a double-stranded
RNA of 18-25 bases, and the cell is contacted with a composition
consisting essentially of the oligomer. In vivo applications of
duplex RNAs are reviewed in Paroo and Corey (2004).
[0048] Typically, the methods of the invention provide at least a
1.2-fold resultant increased synthesis of the target transcript
relative to control conditions and/or basal expression levels. In
other embodiments, increases of at least 1.5, 1.7, 2.0, 2.5, 3.0,
3.5, or 4.0 fold are achieved. Efficient increased synthesis of the
target transcript can be achieved without viral transduction; in
fact, in preferred embodiments the contacting step is free of viral
transduction. While multiple regions of the target promoter can be
targeted, highly efficient increased synthesis of the target
transcript can be achieved with dsRNA targeting just a single
region of the target promoter. For example in one embodiment the
oligomer is a dsRNA of 18-25 bases, there is at least a 2-fold
resultant increased synthesis of the target transcript relative to
control conditions and/or basal expression levels, and a single
region of the target promoter is targeted. Significant increases in
synthesis of the target transcript can be achieved using nanomolar
or picomolar (submicromolar) concentrations of the oligomer, and it
is typically preferred to use the lowest concentration possible to
achieve the desired resultant increased synthesis, e.g. oligomer
concentrations in the 1-100 nM range are preferred; more
preferably, the concentration is in the 1-50 nM, 1-25 nM, 1-10 nM,
or picomolar range.
[0049] As disclosed and exemplified herein, by exploiting a
hitherto unappreciated endogenous mechanism for selective gene
activation, our methods are generally applicable across a wide
variety of target genes, promoter regions, oligomers, mammalian
cell types and delivery conditions. While conditions whereby a
given oligomer selectively activates transcription of a given
target gene are necessarily confirmed empirically (e.g. pursuant to
the protocols described herein), we have consistently found
activating oligomers for every mammalian gene we have studied; and
our data indicate that mammalian cells are generally amenable to
target gene selective activation using these methods.
[0050] In the detecting step of the method, selective increased
synthesis of the target transcript resulting from the oligomer
contacting the cell is detected. This can be determined directly by
measuring an increase in the level of the gene's mRNA transcript,
or indirectly by detecting increased levels of the corresponding
encoded protein compared to controls. Alternatively, resultant
selective increased synthesis of the target transcript may be
inferred based on phenotypic changes that are indicative of
increased synthesis of the target transcript.
[0051] In another aspect of the invention, provided are
polynucleotide oligomers for selectively increasing synthesis of a
target transcript of a gene, the oligomer comprising a nucleotide
sequence of 12-28 bases complementary to a region within a target
promoter of the gene located between nucleotides -100 to +25
relative to a transcription start site of the gene. When introduced
into a cell comprising the gene, the oligomer selectively increases
transcription of the target transcript. In one embodiment, the
target transcript encodes a protein selected from the group
consisting of human major vault protein (MVP), human E-cadherin,
human progesterone receptor (hPR), human p53, and human PTEN. In
further embodiments, the oligomer is RNA, DNA, peptide nucleic acid
or morpholino. In one embodiment, the nucleic acid oligomer is a
dsRNA of 18-25 bases.
[0052] Specific gene targets and dsRNA sequences that selectively
increase transcript synthesis are listed in Table 1. Only one
strand (shown 5' to 3') of each dsRNA is shown. Additionally the
dsRNAs had 3'-dithymidine overhangs on each strand.
TABLE-US-00001 TABLE 1 Gene Target region dsRNA MVP -54 to -36
UGGGCUUGGCCUGCCUUGC (SEQ ID NO: 1) MVP -82 to -64
GGGCCCUUUAACUCCCAAG (SEQ ID NO: 2) E-Cadherin -9 to +10
CCCCCUCUCAGUGGCGUCG (SEQ ID NO: 3) hPR -25 to -6
GGCGUUGUUAGAAAGCUGU (SEQ ID NO: 4) hPR -29 to -10
AGGAGGCGUUGUUAGAAAG (SEQ ID NO: 5) hPR -34 to -15
AGAGGAGGAGGCGUUGUUA (SEQ ID NO: 6) p53 -13 to +6
GCUAAAAGUUUUGAGCUUC (SEQ ID NO: 7) p53 -9 to +10
AAAGUUUUGAGCUUCUCAA (SEQ ID NO: 8) p53 -7 to +12
AGUUUUGAGCUUCUCAAAA (SEQ ID NO: 9) PTEN -13 to +6
CGCGACUGCGCUCAGUUCU (SEQ ID NO: 10) PTEN -9 to +10
ACUGCGCUCAGUUCUCUCC (SEQ ID NO: 11) PTEN -7 to +12
UGCGCUCAGUUCUCUCCUC (SEQ ID NO: 12)
[0053] In a further aspect of the invention, the invention provides
a method of doing business comprising promoting, marketing, selling
or licensing any of the aforementioned inventions.
Example 1
agRNA-Induced Transcriptional Increase
[0054] MVP: Using previously described methods (Janowski et al.,
2005), dsRNAs targeting the major vault protein (MVP; Lange et al,
2000) at -82 to -64 (-82/-64) relative to the transcription start
site, and the p53 site (-54/-36), caused 2.9 and 3.8 fold increases
respectively in MVP expression at the level of RNA and protein.
[0055] E-Cadherin: An RNA targeting the -10/+9 region of E-cadherin
(referred to as EC9), caused a 1.5-2.1 increase in E-cadherin
expression at the level of RNA and protein. RNAs targeting -9/+10,
-13/+6 and -14/+5 caused no increase or inhibited expression and
gene activation. Activation of E-cadherin expression by EC9 was
observed in three independent experiments.
[0056] h-PR: We have tested twenty-one RNAs complementary to
progesterone receptor (PR) (Janowski et al, 2005). Several of these
efficiently blocked gene expression, but in the course of these
experiments we were surprised to note that some RNAs led to small
but reproducible increases in expression. To follow up these
observations we reduced expression of PR to near basal levels by
growing cells in media with reduced levels of serum. When cells
grown under these conditions were transfected with antigene RNAs
(agRNAs), significant increases in hPR expression were observed.
Table 2 shows the region targeted and indicates the level of
increase expression obtained from various experiments.
TABLE-US-00002 TABLE 2 Region targeted Level of Increased
Expression -9/+10 0.5x -11/+8 2.0x -14/+5 1.7x -19/-1 1.9x -22/-3
1.0x -25/-6 1.7 to 4.1x -29/-10 4.5x -34/-15 1.5x to 4.8x -44/-35
1.2x
[0057] The agRNA that targets -9/+10 had previously been shown to
inhibit hPR in cells grown in serum, while all the other RNAs had
been inactive or had shown slight activation. These results are
reproducible and are observed in normal 10% serum (conditions that
activate hPR) and in 2.5% serum (serum-deprived conditions that
lead to a low level of hPR expression).
[0058] p53: While targeting RNAs to the promoter for p53 we
discovered another form of transcriptional modulation. Expression
of the major p53 isoform was decreased (abolished), while
expression of a p53 isoform having a lower apparent molecular
weight was increased when we targeted the following regions:
-7/+12, -9/+10, and -13/+6 relative to the transcription start site
of the major isoform of p53 (see Table 1, SEQ ID NOs 7-9). This
lower molecular weight p53 isoform was also recently described by
others (Bourdon, et al, 2005; Rohaly et al, 2005). Altered RNA
expression was confirmed by RT-PCR.
[0059] The p53 gene promoter contains alternative transcription
start sites. Table 3 discloses exemplary p53 transcription start
site proximate target region/oligo pairs for selectively increasing
target transcript synthesis. Only one strand (shown 5' to 3') of
each dsRNA is shown.
TABLE-US-00003 TABLE 3 Gene Target region dsRNA p53.sup.1 -13 to +6
UGACUCUGCACCCUCCUCC (SEQ ID NO: 13) p53.sup.1 -9 to +10
UCUGCACCCUCCUCCCCAA (SEQ ID NO: 14) p53.sup.1 -7 to +12
UGCACCCUCCUCCCCAACU (SEQ ID NO: 15) p53.sup.2 -13 to +6
AUUACUUGCCCUUACUUGU (SEQ ID NO: 16) p53.sup.2 -9 to +10.
CUUGCCCUUACUUGUCAUG (SEQ ID NO: 17) p53.sup.2 -7 to +12
UGCCCUUACUUGUCAUGGC (SEQ ID NO: 18) .sup.1Bourdon et al., 2005
.sup.2Lamb and Crawford, 1986
[0060] We have also observed similar upregulation of isoform
expression of a second gene upon transfection of cells with duplex
RNAs that target PTEN (see Table 1, SEQ ID NOs 10-12).
Example 2
Increased Human Progesterone Receptor (hPR) Expression by Antigene
PNA (agPNA) Oligomers Targeting Near the Transcription Start
Site
[0061] Cell Culture T47D breast cancer cells (American Type Cell
Culture Collection, ATCC) are maintained at 37.degree. C. and 5%
CO.sub.2 in RPMI media (ATCC) supplemented with 10% (v/v)
heat-inactivated (56.degree. C., 1 hr) fetal bovine serum (Gemini
Bioproducts), 0.5% non-essential amino acids (Sigma), 0.4 units/mL
bovine insulin (Sigma) and 100 units/ml penicillin and 0.1 mg/ml
streptomycin (Sigma).
[0062] Lipid-Mediated Transfection of PNA PNAs are obtained as
described (Kaihatsu et al, 2004). Two days before transfection (day
2), T47D cells are plated at 80,000 cells per well in 6-well plates
(Costar). On the day of transfection (day 0) duplexes (200 nM) and
Oligofectamine (9 .mu.l per well, Invitrogen) is diluted in Optimem
(Invitrogen) according to the manufacturers' instructions. Media is
changed 24 h later (day 1). On day 3 cells are passaged 1:4 into
new 6-well plates. Cells are transfected a second time on day 5.
Cells are harvested day 8. hPR protein levels are evaluated by
Western analysis using anti-hPR antibody (Cell Signaling
Technologies).
[0063] RNA Analysis. Total RNA from treated T47D cells is extracted
using trizol (TRIzol, Invitrogen). RNA is treated with
deoxyribonuclease to remove contaminating DNA, and 4 .mu.g are
reverse transcribed by random primers using Superscript II RNase
H-reverse transcriptase (Invitrogen).
[0064] Microscopy. Cells are imaged by confocal microscopy using a
Zeiss Axiovert 200 M inverted transmitted light microscope (Carl
Zeiss Microimaging). Approximations of cell height are made by
tracking distances in the Z-plane using an automated program.
Individual cells are chosen for observation and then the microscope
is underfocused until no part of the individual cell is in focus.
The underfocus position in the Z-plane is noted and then the focal
plane is moved upward through the cell until it is completely out
of focus. The overfocus position is noted and a crude estimate of
the height (Zdimension) of the cell can be calculated.
[0065] Cellular Uptake of Biologically Active PNAs PNAs are
introduced into cells by complexing them with partially
complementary DNA oligonucleotides and cationic lipid. The lipid
promotes internalization of the DNA, while the PNA enters as cargo
and is subsequently released.
[0066] Activation of hPR Expression by agPNAs. 19-base PNAs
targeting near the transcription start site (-100 to +25) of hPR,
and contain C- and N-terminal lysines are prepared and transfected
into cells at a concentration of 200 nm. AgPNA induced increase of
hPR protein expression is measured by Western analysis.
Example 3
VEGF-Activating agRNAs Increase Vascularization
[0067] The promoter region of the human VEGF gene has been
characterized (see e.g. Tischer et al, 1991). The transcription
start site is at position 2363 in the published sequence (GenBank
Accession no. AF095785.1). 19-mer agRNAs fully complementary to the
template strand and targeting near the transcription start site of
the gene (-50 to +25, where transcription start is +1) are
prepared; exemplary agRNAs are shown in Table 4 (second strand and
dinucleotide overhangs not shown).
TABLE-US-00004 TABLE 4 agRNA Sequence Location hV2
GAUCGCGGAGGCUUGGGGC -2/+17 (SEQ ID NO: 19) hV6 GGAGGAUCGCGGAGGCUUG
-6/+13 (SEQ ID NO: 20) hV7 GGGAGGAUCGCGGAGGCUU -7/+12 (SEQ ID NO:
21) hV8 GGGGAGGAUCGCGGAGGCU -8/+11 (SEQ ID NO: 22) hV9
CGGGGAGGAUCGCGGAGGC -9/+10 (SEQ ID NO: 23) hV10 GCGGGGAGGAUCGCGGAGG
-10/+9 (SEQ ID NO: 24) hV11 AGCGGGGAGGAUCGCGGAG -11/+8 (SEQ ID NO:
25) hV12 UAGCGGGGAGGAUCGCGGA -12/+7 (SEQ ID NO: 26) hV13
GUAGCGGGGAGGAUCGCGG -13/+6 (SEQ ID NO: 27) hV14 GGUAGCGGGGAGGAUCGCG
-14/+5 (SEQ ID NO: 28) hV15 UGGUAGCGGGGAGGAUCGC -15/+4 (SEQ ID NO:
29) hV19 UCGGCUGGUAGCGGGGAGG -19/-1 (SEQ ID NO: 30) hV24
AAAAGUCGGCUGGUAGCGG -24/-6 (SEQ ID NO: 31) hV25 UAAAAGUCGGCUGGUAGCG
-25/-7 (SEQ ID NO: 32) hV30 UUUUUAAAAGUCGGCUGGU -30/-12 (SEQ ID NO:
33) hV35 UUUUUUUUUUAAAAGUCGG -35/-17 (SEQ ID NO: 34) hV40
CCCCCUUUUUUUUUUAAAA -40/-22 (SEQ ID NO: 35) hV45
CGCCCCCCCCUUUUUUUUU -45/-27 (SEQ ID NO: 36) hV49
CAUGCGCCCCCCCCUUUUU -49/-31 (SEQ ID NO: 37)
[0068] The effect of the agRNAs on selectively increasing synthesis
of VEGF transcripts is determined in primary human umbilical vein
cells (HUVECs). Resultant selective increased synthesis of the VEGF
transcript is detected inferentially from increases in cell
proliferation and/or directly by measuring increases in VEGF gene
transcripts relative to controls. agRNAs resulting in at least a
2-fold increase in VEGF gene transcription are evaluated in animal
model and clinical studies for treatment of myocardial ischemia as
described below.
[0069] Ischemic Heart Model Adenoviral vectors are constructed for
of VEGF-activating agRNAs to an ischemic heart mouse model using
known methods (see e.g. Zender et al, 2003; Su et al, 2002; and Su
et al, 2004). CD1 mice (Charles River Breeding Laboratories) are
anesthetized with 15-16 .mu.l of 2.5% Avertin per gram of body
weight by i.p. injection. After the respiration of the animal is
controlled by a Small Animal Volume Controlled Ventilator (Harvard
Rodent Ventilator, model 683, South Natick, Mass.), a thoracotomy
incision is made in the fourth intercostal space. A surgical
retractor is put in the incision to expose the heart. The anterior
descending coronary artery is ligated permanently with a 6-0
nonabsorbable surgical suture to induce ischemia. 1.times.10.sup.11
genomes of viral vectors in 50 .mu.l of Hepes saline (pH 7.4) is
injected directly to multiple sites of the myocardium on the left
ventricle wall around the ischemic region. Control mice receive
buffer injections. Cardiac function is assessed 4 weeks after the
surgery. Left ventricular end diastolic dimension (LVDd) and end
systolic dimension (LVDs) are measured. The percentage of
fractional shortening (FS %) is calculated as
(LVDd-LVDs)/LVDd.times.100.
[0070] Hearts collected after echocardiography are sectioned and
stained with anti-platelet endothelial cell adhesion molecule 1 and
smooth muscle .alpha.-actin antibodies. Vessels are counted on six
areas, three on the anterior wall and three on the posterior wall
in cross sections of the left ventricle. Area 1 is made up entirely
of muscle tissue, area 2 has both muscle and scar, and area 3 has
scar only. Vectors are injected into area 2 at the anterior wall.
Hence, comparison between the injected areas in the anterior and
the corresponding uninjected posterior areas indicates the effect
of the agRNA on VEGF expression. Capillary density is expressed as
the ratio of capillary to cardiac myocyte for area 1 and as the
number of capillaries per mm.sup.2 for areas 2 and 3. The density
of .alpha.-actin-positive vessels is expressed as the number of
vessels per mm.sup.2 or all areas. Activation of VEGF expression is
demonstrated by an increase in capillaries and
.alpha.-actin-positive vessels in all three areas of the anterior
walls compared with the posterior walls in the same hearts and
compared with the anterior walls of control groups.
[0071] Clinical Trials: The safety and efficacy of VEGF-activating
agRNA therapy in humans is evaluated in a clinical study designed
after a study described by Losordo et al (2002). Eligible patients
include Canadian Cardiovascular Society (CCS) class III or IV
angina refractory to maximum medical therapy, multivessel coronary
artery disease not suitable for bypass surgery or angioplasty, and
reversible ischemia on stress SPECT Tc 99m sestamibi nuclear
imaging. Subjects are excluded if they had a previous history or
current evidence of malignancy, active diabetic retinopathy, or
evidence of severe LV systolic dysfunction (LV ejection fraction
[EF]<20% by transthoracic 2D echocardiography).
[0072] VEGF-inducing agRNA-expressing vectors are injected into the
patients. Subjects undergo nonfluoroscopic LV EMM immediately
before injection of the vector to guide injections to foci of
ischemic myocardium. Follow-up EMM is performed at 12 weeks after
injections. The pre-specified primary efficacy parameters are
change from baseline in CCS angina classification and exercise
tolerance at the 12-week follow-up visit.
Example 4
agRNA-Induced Transcriptional Increase
[0073] We reasoned that gene activation could be more readily
observed against a low basal level of gene expression. Therefore,
to address our hypothesis, we introduced duplex RNAs into MCF-7
cells, a breast cancer cell line with a much lower basal level of
PR protein expression than observed in T47D cells (Janowski et al
(2006a) Nature Struc. Mol. Biol. 13:787-792; Jenster et al (1997)
Proc Natl Acad Sci USA 94:7879-7884).
[0074] We initiated testing with RNA PR11, a duplex complementary
to the PR promoter sequence from -11 to +8. We chose PR11 because
it had not inhibited PR expression in T47D cells but was surrounded
by agRNAs that were potent inhibitors. For comparison, we also
tested RNAs PR9 and PR26 that we had previously shown to be potent
inhibitors of PR expression in T47D cells.
[0075] We introduced duplex RNA PR11 into MCF-7 cells using
cationic lipid (Janowski et al (2006b) Nature Protocols 1:436-443)
and observed an 18-fold increase in levels of PR protein by Western
analysis, indicating that agRNAs could produce substantial
up-regulation of gene expression when tested in an appropriate
cellular context. Addition of PR9 did not affect PR expression,
while PR26 yielded a modest 2-fold increase in PR levels. Two
siRNAs that were complementary to downstream coding sequences
within PR mRNA inhibited expression of PR protein, demonstrating
that PR levels could be reduced by standard post-transcriptional
silencing in MCF-7 cells.
[0076] After observing RNA-mediated activation of gene expression
by PR11 we assayed the specificity and potency of the phenomenon.
We tested a battery of mismatch and scrambled control duplexes,
including mismatches that preserved complementarity at the either
end of the duplex. These control duplexes did not increase
expression of PR, demonstrating that upregulation was
sequence-specific. Addition of PR11 at varied concentrations
demonstrated that activation was potent, with 17-fold activation
achieved at a 12 nM concentration.
[0077] We then re-examined gene activation by duplex RNAs in T47D
cells. To facilitate unambiguous observation of activation, we
reduced the basal level of PR expression by growing the cells in
culture medium containing charcoal-treated serum (Hurd et al (1995)
J. Biol. Chem.). As expected, use of serum-stripped media lacking
hormones reduced PR expression. Addition of RNA PR11 induced PR
expression to levels observed for T47D cells in normal media. These
results demonstrate that PR11 has the same physiologic effect in
two different breast cancer cell types and that PR11 is able to
counteract a well-established mechanism for manipulating hormone
receptor expression.
[0078] PR protein is expressed as two isoforms, PR-A and PR-B,
which play differing roles in physiologic processes (Conneely et
al, (2003) Mammary Gland Biol. Neoplasia 8:205-214). The promoter
for PR-B is upstream from the promoter for PR-A and the RNAs used
in this study target the PR-B promoter. We had previously observed
that agRNAs, siRNAs, antisense PNAs, or antigene PNAs that target
the PR-B promoter (agRNA, antigene PNA) or PR-B mRNA (siRNA,
antisense PNA) also reduce levels of PR-A (Janowski et al, 2005;
Janowski et al, 2006a; Janowski et al, 2006b; and Janowski et al
(2006c) Nature Chem. Biol 1:210-215) indicating that expression of
PR-A is linked to expression of PR-B. We now observe that RNAs
targeting the PR-B promoter can also enhance expression of both
PR-B and PR-A protein, providing complementary evidence that
expression of the isoforms is linked.
[0079] To correlate activity with target sequence, we tested a
series of duplex RNAs targeted to sequences throughout the region
-56 to +17 within the PR promoter. Several of these duplex RNAs
induced expression of PR by 5 fold or greater (Table 5). Small
shifts in target sequence had large consequences for activation.
For example, a single base shift upstream (PR12) or downstream
(PR10) from PR11 substantially reduced activation. Experiments were
repeated several times with similar results. These data indicate
that sequences throughout the promoter are suitable targets and
that the requirements for RNA-mediated gene activation are
flexible.
TABLE-US-00005 TABLE 5 RNA Targeting PR Fold Activation Relative to
Mismatch Controls -2/+17 8x -6/+13 6x -9/+10 7x -10/+9 1x -11/+8
19x -12/+7 3x -13/+6 4x -14/+5 16x -19/-1 7x -22/-3 13x -23/-4 6x
-24/-5 3x -25/-6 8x -26/-7 10x -29/-10 6x -39/-20 3x -49/-30 3x
[0080] We performed order of addition experiments in which inactive
RNAs PR10 or PR12 were transfected either before or after
transfection with activating RNA PR11 (Table 6). When PR10 or PR12
were added to cells first, we observed that subsequent addition of
PR11 did not result in activation. When PR11 was added to cells
first, PR10 or PR12 did not block gene activation. These
competition assays indicate that inactive RNAs PR10 and PR12 bind
at the same target sequence as PR11. Recognition is sufficient to
block binding of PR11 and prevent activation of PR expression.
Competition of PR11 with PR10 and PR12 further documents the
target- and sequence-specificity of RNA-mediated activation of
PR.
TABLE-US-00006 TABLE 6 Transfection 1 Transfection 2 Outcome
Activating RNA PR11 Inactive RNA PR8 Activation Activating RNA PR11
Inactive RNA PR12 Activation Inactive RNA PR12 Activating RNA PR11
No Activation Inactive RNA PR8 Activating RNA PR11 No
Activation
[0081] To determine whether duplex RNAs could activate expression
of other genes we examined a series of RNAs targeted to major vault
protein (MVP) (Huffman and Corey, (2004) Biochemistry
44:2253-2261). We chose MVP because we previously silenced its
expression with agRNAs (Janowski et al, 2005). MVP6 and MVP9
inhibited gene expression, a result that we had reported previously
(Janowski et al, 2005). By contrast, MVP35 (corresponding to
nucleotides 1819-1837 of Genbank Accession no. AJ238509,
GI:583487), MVP54, and MVP82, increased expression by 2-4 fold
above normal levels. These data indicate that duplex RNAs can
enhance expression of genes with relatively high basal levels of
expression, similar to our initial observation of RNA-mediated
upregulation of PR in T47D cells.
[0082] Quantitative PCR (QPCR) reveals that treatment of MCF-7
cells with PR11 enhances expression of PR mRNA under a variety of
cell culture conditions. We had previously shown that inhibition of
PR expression in T47D cells by siRNAs (Hardy et al (2006) Mol
Endocrinol, Epub ahead of print Jun. 13, 2006) or agRNAs
(unpublished) significantly increases expression of
cyclooxygenase-2 (COX-2) after induction with interleukin1 beta
(IL-113). We now observe that activation of PR gene expression in
MCF-7 cells after treatment with RNA PR11 reduces COX-2 expression
in the presence or absence of IL-1.beta.. Treatment of cells with
PR11 did not alter levels of estrogen receptor-alpha (ER-alpha), a
key regulator of PR expression. Accordingly, a specific embodiment
of our invention is a method for decreasing Cox-2 expression in a
cell by contacting the cell with a polynucleotide oligomer of 12-28
bases complementary to a region located between nucleotides -100 to
+25 relative to a transcription start site of the human
progesterone receptor (hPR) gene under conditions whereby the
oligomer selectively increases synthesis of the hPR; and detecting
decreased synthesis of the Cox-2; wherein the oligomer is
preferably double-stranded RNA.
[0083] Our data demonstrate that activating RNAs can be used to
manipulate expression of physiologically-relevant downstream target
genes in a predictable manner and that the induced PR is fully
functional.
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(2000) Proc. Natl. Acad. Sci. USA, 97: 5633-5638.
Sequence CWU 1
1
37119RNAartificialantigene RNA polynucleotide oligomer 1ugggcuuggc
cugccuugc 19219RNAartificialantigene RNA polynucleotide oligomer
2gggcccuuua acucccaag 19319RNAartificialantigene RNA polynucleotide
oligomer 3cccccucuca guggcgucg 19419RNAartificialantigene RNA
polynucleotide oligomer 4ggcguuguua gaaagcugu
19519RNAartificialantigene RNA polynucleotide oligomer 5aggaggcguu
guuagaaag 19619RNAartificialantigene RNA polynucleotide oligomer
6agaggaggag gcguuguua 19719RNAartificialantigene RNA polynucleotide
oligomer 7gcuaaaaguu uugagcuuc 19819RNAartificialantigene RNA
polynucleotide oligomer 8aaaguuuuga gcuucucaa
19919RNAartificialantigene RNA polynucleotide oligomer 9aguuuugagc
uucucaaaa 191019RNAartificialantigene RNA polynucleotide oligomer
10cgcgacugcg cucaguucu 191119RNAartificialantigene RNA
polynucleotide oligomer 11acugcgcuca guucucucc
191219RNAartificialantigene RNA polynucleotide oligomer
12ugcgcucagu ucucuccuc 191319RNAartificialantigene RNA
polynucleotide oligomer 13ugacucugca cccuccucc
191419RNAartificialantigene RNA polynucleotide oligomer
14ucugcacccu ccuccccaa 191519RNAartificialantigene RNA
polynucleotide oligomer 15ugcacccucc uccccaacu
191619RNAartificialantigene RNA polynucleotide oligomer
16auuacuugcc cuuacuugu 191719RNAartificialantigene RNA
polynucleotide oligomer 17cuugcccuua cuugucaug
191819RNAartificialantigene RNA polynucleotide oligomer
18ugcccuuacu ugucauggc 191919RNAartificialantigene RNA
polynucleotide oligomer 19gaucgcggag gcuuggggc
192019RNAartificialantigene RNA polynucleotide oligomer
20ggaggaucgc ggaggcuug 192119RNAartificialantigene RNA
polynucleotide oligomer 21gggaggaucg cggaggcuu
192219RNAartificialantigene RNA polynucleotide oligomer
22ggggaggauc gcggaggcu 192319RNAartificialantigene RNA
polynucleotide oligomer 23cggggaggau cgcggaggc
192419RNAartificialantigene RNA polynucleotide oligomer
24gcggggagga ucgcggagg 192519RNAartificialantigene RNA
polynucleotide oligomer 25agcggggagg aucgcggag
192619RNAartificialantigene RNA polynucleotide oligomer
26uagcggggag gaucgcgga 192719RNAartificialantigene RNA
polynucleotide oligomer 27guagcgggga ggaucgcgg
192819RNAartificialantigene RNA polynucleotide oligomer
28gguagcgggg aggaucgcg 192919RNAartificialantigene RNA
polynucleotide oligomer 29ugguagcggg gaggaucgc
193019RNAartificialantigene RNA polynucleotide oligomer
30ucggcuggua gcggggagg 193119RNAartificialantigene RNA
polynucleotide oligomer 31aaaagucggc ugguagcgg
193219RNAartificialantigene RNA polynucleotide oligomer
32uaaaagucgg cugguagcg 193319RNAartificialantigene RNA
polynucleotide oligomer 33uuuuuaaaag ucggcuggu
193419RNAartificialantigene RNA polynucleotide oligomer
34uuuuuuuuuu aaaagucgg 193519RNAartificialantigene RNA
polynucleotide oligomer 35cccccuuuuu uuuuuaaaa
193619RNAartificialantigene RNA polynucleotide oligomer
36cgcccccccc uuuuuuuuu 193719RNAartificialantigene RNA
polynucleotide oligomer 37caugcgcccc ccccuuuuu 19
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